Method, microreactor and apparatus for carrying out real-time nucleic acid amplification

ABSTRACT

A method for carrying out nucleic acid amplification, includes providing a reaction chamber ( 31 ), accommodating an array ( 36 ) of nucleic acid probes ( 37 ) at respective locations, for hybridizing to respective target nucleic acids; and introducing a solution ( 50 ) into the reaction chamber ( 31 ), wherein the solution ( 50 ) contains primers, capable of binding to target nucleic acids, nucleotides, nucleic acid extending enzymes and a sample including nucleic acids. The a structure of the nucleic acid probes ( 37 ) and of the primers so that a hybridization temperature (T H ) of the probes ( 37 ) is higher than an annealing temperature (T A ) of the primers, whereby hybridization and annealing take place in respective separate temperature ranges (R H , R A ).

TECHNICAL FIELD

The present invention relates to a method, to a microreactor and to anapparatus for carrying out real-time nucleic acid amplification. Inparticular, amplification process exploits an array of oligonucleotideprobes.

BACKGROUND ART

Typical procedures for analyzing biological materials, such as nucleicacid, protein, lipid, carbohydrate, and other biological molecules,involve a variety of operations starting from raw material. Theseoperations may include various degrees of cell separation orpurification, cell lysis, amplification or purification, and analysis ofthe resulting amplification or purification product.

As an example, in DNA-based blood analyses samples are often purified byfiltration, centrifugation or by electrophoresis so as to eliminate allthe non-nucleated cells, which are generally not useful for DNAanalysis. Then, the remaining white blood cells are broken up or lysedusing chemical, thermal or biochemical means in order to liberate theDNA to be analyzed. Next, the DNA is denatured by thermal, biochemicalor chemical processes and amplified by an amplification reaction, suchas PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA(strand displacement amplification), TMA (transcription-mediatedamplification), RCA (rolling circle amplification), and the like. Theamplification step allows the operator to avoid purification of the DNAbeing studied because the amplified product greatly exceeds the startingDNA in the sample.

If RNA is to be analyzed the procedures are similar, but more emphasisis placed on purification or other means to protect the labile RNAmolecule. RNA is usually copied into DNA (cDNA) and then the analysisproceeds as described for DNA.

Finally, the amplification product undergoes some type of analysis,usually based on sequence or size or some combination thereof. In ananalysis by microarray hybridization, for example, the amplified DNA ispassed over a plurality of detectors made up of individualoligonucleotide detector fragments that are anchored, for example, onelectrodes. If the amplified DNA strands are complementary to theoligonucleotide detectors or probes, stable bonds will be formed betweenthem (hybridization) under specific temperature conditions. Thehybridized detectors can be read by observation using a wide variety ofmeans, including optical, electromagnetic, electromechanical or thermalmeans.

Other biological molecules are analyzed in a similar way, but typicallymolecule purification is substituted for amplification, and detectionmethods vary according to the molecule being detected. For example, acommon diagnostic involves the detection of a specific protein bybinding to its antibody. Such analysis requires various degrees of cellseparation, lysis, purification and product analysis by antibodybinding, which itself can be detected in a number of ways. Lipids,carbohydrates, drugs and small molecules from biological fluids areprocessed in similar ways. However, we have simplified the discussionherein by focusing on nucleic acid analysis, in particular DNA analysis,as an example of a biological molecule that can be analyzed using thedevices of the invention.

PCR is time consuming, because it is necessary to perform 20-30iterations of the basic thermal cycle to ensure that any target nucleicacid has been sufficiently amplified so as to be detectable. Further,the amplification and detection reactions are often sequential, furtherconsuming valuable time.

DISCLOSURE OF INVENTION

The object of the invention is to provide a method, a microreactor andan apparatus for carrying out real time nucleic acid amplification thatis free from the above described limitations.

According to the present invention, a method, a microreactor and anapparatus for carrying out nucleic acid amplification are provided, asclaimed in claims 1, 9 and 10, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

For the understanding of the present invention, some embodiments thereofwill be now described, purely as non-limitative examples, with referenceto the enclosed drawings, wherein:

FIG. 1 is a system depiction of an apparatus for carrying out nucleicacid amplification according to one embodiment of the present invention;

FIG. 2 is a top plan view of a microreactor according to one embodimentof the present invention;

FIG. 3 is a cross-sectional view of the microreactor of FIG. 2, takenalong the line III-III of FIG. 2;

FIG. 4 is a plot showing absorption and emission spectra of a dye-DNAcomplex; and

FIG. 5 is a temperature profile used in a method according to oneembodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

With reference to FIG. 1, a biochemical analysis apparatus 1 comprises acomputer system 2, a temperature control module 3, a reader device 4,and a microreactor 5 for performing biochemical analyses, that isprovided on a board 6 to form a disposable microreactor cartridge 7.

The microreactor cartridge 7 is loadable into a receptacle 9 of theapparatus 1 and is provided with an interface 8 for coupling with thetemperature control module 3 and the reader device 4.

The temperature control module 3 and the reader device are bothcontrolled by a processing unit 10 of the computer system 2.

The temperature control module 3 includes a temperature controller 11and a power source 12. The temperature controller 11 is configured toreceive a temperature signal S_(T) from a temperature sensor (describedlater on and here not shown) on the microreactor cartridge 7. Thetemperature control module 3 may also include a cooling element 13, e.g.a Peltier module or a fan coil, which is controlled by the temperaturecontroller 11 and is thermally coupled to the microreactor 5 when thecartridge 7 is loaded in the reader device 4. The power source 12 andthe cooling element 13 are operable by the temperature controller 11respectively to deliver power to heaters (also described later on andhere not shown) coupled to the microreactor 5 and to cool the cartridge7, in order to set an operating temperature in accordance with atemperature profile (see e.g., FIG. 5).

In one embodiment, the reader device 4 is configured to perform opticaldetection of the reaction products in the microreactor 5, as hereinafterdescribed. In particular, the reader device 4 includes a light source 15for illuminating the microreactor 5 with light at an excitationwavelength; and an image detector 16, configured to receive fluorescenceradiation emitted from the microreactor 5, in response to the light atthe excitation wavelength. However, it is understood that other ways tocarry out detection are available and can be exploited, in place ofoptical detection. For example electrochemical detection can beperformed.

FIGS. 2 and 3 illustrate in detail one embodiment of the microreactor 5.However, it is understood that the configuration of the microreactor 1herein illustrated and described can not be considered in any waylimiting and numerous different configurations could be exploited aswell.

The microreactor 5 comprises a body 30, having a recess wherein areaction chamber 31 is formed.

The body 30 includes a substrate 32, covered with a stack of layersincluding, in one embodiment, a dielectric layer 33, a passivation layer34 and a structural layer 35. The reaction chamber 31 is formed in thestructural layer 35 and is downwardly delimited by the passivation layer34.

A microarray 36 of DNA probes 37 is accommodated in the reaction chamber31, while heaters 38 and a temperature sensor 39 are embedded in thepassivation layer 34. Probes include a variety of short oligonucleotidesequences that are placed at specific locations of the microarray 36.

The substrate 32 is made of a thermally conductive material, such asundoped silicon.

In one embodiment, the dielectric layer 33 is of silicon dioxide and hasa thickness of e.g. 0.1 μm to 1 μm. The heaters 38 and the temperaturesensor 39 are arranged on the dielectric layer 33 and therefore they areelectrically insulated from the substrate 32. However, due to the smallthickness of the dielectric layer 33, the heaters 38 and the temperaturesensor 39 are thermally coupled to the substrate 32. Moreover, theheaters 38 and the temperature sensor 39 are electrically connected torespective pads 40, for coupling with the temperature control module 3and reader device 4 through the interface 8 of the cartridge 7. When thecartridge 7 is loaded into the receptacle 9 (FIG. 1), the heaters 38 areconnected to the power source 12 for receiving electrical power and thetemperature sensor 39 is connected to the temperature controller unit 11for providing a temperature signal S_(T).

The passivation layer 34, also of silicon dioxide, is arranged betweenthe dielectric layer 13 and the structural layer 35 and incorporates theheaters 38 and the temperature sensor 39. A top surface of thepassivation layer 34 defines a bottom wall of the reaction chamber 31.

The structural layer 35, e.g. of silicon or glass, is bonded to thepassivation layer 34 and has an opening therein defining the reactionchamber 31. The design of the heaters 38 may be optimized according toindividual configurations of the reaction chamber 31, in order toachieve desired temperature profiles. The temperature sensor 39 ispreferably arranged under the reaction chamber 31.

The reaction chamber 31 is closed by a transparent cap layer 46 (notshown in FIG. 2) (not labeled in 3 either?), attached or bonded to thestructural layer 35, and which may have a small opening 43 forintroducing samples to the reaction chamber 31.

The microarray 36 comprises a plurality of nucleic acid probes 37,preferably single strand deoxy-oligonucleotides, grafted to thepassivation layer 34 at respective locations. Probes 37 are designed tohybridize to target DNA at a specific hybridization temperature when areaction, such as nucleic acid amplification, is carried out in thereaction chamber 31.

Hereinafter reference will be made to a nucleic acid analysis processincluding PCR (Polymerase Chain Reaction). As is known, PCR is acyclical process involving a series of enzyme-mediated reactions whosefinal result are identical copies of the target nucleic acid. A rawbiological sample is preliminarily processed by conventional steps ofcell separation or purification and cell lysis. Then, the sample isadded to a solution comprising enzymes (typically a DNA polymerase suchas TAQ), primers, the four nucleotides (collectively referred to asdNTPs), cofactor, buffer, and a fluorescent dye capable of binding todouble-helix DNA. Such dyes include, but are not limited, bisbenzimideor indole-derived stains (Hoechst 33342, Hoechst 33258 and4′,6-diamidino-2-phenylindole), phenanthridinium stains (ethidiumbromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1iodide, SYBR Green I and SYBR Gold). The fluorescent dye is preferablyselected from the group of cyanine dyes and, in one example, is SYBRGreen I. As illustrated in FIG. 4, a dye-DNA complex, that forms duringthe amplification process, adsorbs visible radiation AVR selectivelyaround a wavelength of 488 nm (blue) and emits visible radiation EVRwith a maximum of emission at 522 nm (green).

The sequences of the probes 37 and of the primers determine ahybridization temperature, at which the probes hybridize tocomplementary target DNA single strands, and an annealing temperature,at which the primers bind to their complementary sequences on the targetDNA. In one embodiment, the probes 37 and the primers are selected suchthat the hybridization temperature is higher than the annealingtemperature, so that hybridization and annealing take place in separateand spaced apart (non overlapping) temperature ranges. Thus, at thehybridization temperature, primer annealing is prevented and at theannealing temperature the primers are allowed to bind to denaturedtarget DNA.

Apart from parameters of the solution (such as salinity), thehybridization and annealing temperatures are determined by the sequenceof the probes 37 and of the primers. Namely, for complementary DNAstrands, the highest hybridization rate is achieved at a temperature ofabout 20-25° C. below the melting temperature of the DNA helix. Forprobes 37, that are composed of short nucleotide sequences, thehybridization temperature is about 5-10° C. below the meltingtemperature.

Thus, once the composition of the solution has been defined, thehybridization temperature may be determined by setting the meltingtemperature of the probe-target DNA pair. The melting temperature of ahybrid in a solution, such as a probe-target DNA pair, is given by

T_(M)=81.5+16.6(log M)+0.41(% G+C)—0.61(% form)-500/L

where T_(M) is the melting temperature, M is the molarity of univalentcations, (% G+C) is the percentage of guanine and cytosine, (% form) isthe percentage of formamide and L is the coupling length, i.e. thelength of the sequence in terms of number of paired bases.

If coupling length L is less than 50, however, the melting temperatureis preferably determined from the equation:

T _(M)=2N _(AT)+4N _(GC)

where N_(AT) is the number of A-T (adenine-thymine) pairs and N_(GC) isthe number of G-C (guanine-cytosine) pairs in the sequence.

In both cases, however, the melting temperature T_(M) in a givensolution is essentially determined by the number of G-C pairs and by thecoupling length L. Thus, the sequence of the probes 37 and of theprimers, namely the number of G-C pairs and the coupling length L, canbe selected to set the hybridization temperature and the annealingtemperature such that annealing is prevented during hybridization andvice versa.

By way of example, a primer may be defined by a sequence of 19 dNTPs (tohave a corresponding coupling length of 19), capable of forming 9 A-Tpairs and 10 G-C pairs upon annealing. Once a salinity of the solutionhas been defined, the primer melting temperature T_(MPRIMER) can becalculated. If the primer melting temperature T_(MPRIMER) is determinedto be e.g. 57° C., the annealing temperature is about 52° C.(approximately 5° C. less than the primer melting temperatureT_(MPRIMER)). A probe 37 having 50 bases may be selected from a sequencewith high content of guanine and cytosine, to have high probe meltingtemperature T_(MPROBE) and hybridization temperature. In one example,the probe 37 may be defined by a sequence of 50 dNTPs (to have acorresponding coupling length of 50) and contains 54% ofguanine-cytosine and 46% of adenine-thymine. In the same solution, theprobe melting temperature T_(MPROBE) of the described probe 37 would be85° C. and the hybridization temperature about 80° C. (again, 5° C. lessthan the probe melting temperature T_(MPROBE).) Thus, hybridization andannealing take place at separate temperature intervals and areselectively and exclusively carried out. In other words, when theprimers can bind to target DNA, the probes 37 cannot hybridize and, viceversa, when the temperature conditions allow hybridization, annealing isprevented. At the annealing temperature, the probes 37 are not activeand do not interact with primers. Hence, the hybridization of the probes37 can be avoided, although the DNA amplification process is carried outin the same reaction chamber 31 accommodating the microarray 36.

To start an analysis process involving PCR, a solution 50, obtained asdescribed above, is supplied to the reaction chamber 31 and themicroreactor 5 is loaded into the receptacle 9 of the apparatus 1.

DNA amplification by PCR is then carried out in the reaction chamber 31.To this end, the temperature controller 11 operates the power source 12and the cooling element 13 to controllably deliver electric power W_(E)to the heaters 38 and to cyclically control an operating temperatureT_(O) of the solution 50 in the reaction chamber 31 in accordance with adesired amplification temperature profile.

An example of an amplification temperature profile for the operatingtemperature T_(O) during a PCR amplification cycle is shown in FIG. 5.PCR amplification cycles are iteratively carried out until stopconditions are met. A hybridization range R_(H) and an annealing rangeR_(A), separate and non overlapping, are also illustrated in FIG. 5.

Double stranded DNA is first denatured at a denaturation temperatureT_(D) (94° C. for 10 s to 60 s). In this step, DNA helixes separate intosingle strands.

Then, the solution is set at about a hybridization temperature T_(H) ofthe hybridization range R_(H), at which the hybridization rate of theprobes 37 is maximum. Selective hybridization of the probes 37 to anycomplementary target DNA is thus carried out. The hybridizationtemperature T_(H) of the probes 37 is determined as above explained andis lower than the denaturation temperature T_(D). With reference to theprobe sequence and solution composition already referred to, thehybridization temperature T_(H) is about 80° C. At the hybridizationtemperature T_(H), the probes 37 of the microarray 36 are available forbinding to complementary DNA strands provided in the solution 50.Annealing of the primers, however, does not take place at thistemperature.

Optical detection of hybridized probes 37 is carried out as long as thehybridization temperature T_(H) is maintained in the reaction chamber31. Fluorescent dyes contained in the solution 30 bind to hybridizedprobes 37. Thus, hybridized probes 37 respond to a confocal lightstimulation from a source at a source wavelength (488 nm in the exampledescribed) and emit visible radiation at an emission wavelength (522nm), greater than the source wavelength.

After first denaturation, hybridization and detection, double strandedDNA is again denatured at the denaturation temperature T_(D). The firstand second denaturation may have the same duration, in one embodiment.Fluorescent molecules are then released in the solution after the seconddenaturation, due to separation of target DNA from the respective probes37.

Then, the solution 50 in the reaction chamber 31 is cooled to about anannealing temperature T_(A) in the annealing range R_(A), that in oneembodiment may be of 50° C. to 70° C. (for 10 s to 60 s). In oneembodiment, in particular, the annealing temperature T_(A) is 68°. Atthis stage, primers, which are more numerous than the probes,effectively compete for and bind to their complementary sequences in thefloating target DNA thus allowing amplification of target sequences.

Finally, the solution is heated to an extension temperature T_(E), atwhich DNA polymerase extends primers, by adding nucleotides that arecomplementary to the target strand. The extension temperature T_(E) isintermediate between the annealing temperature T_(A) and thehybridization temperature T_(H) (e.g. 72° C., for 10 s to 60 s). Duringextension, the fluorescent molecules can bind the PCR product, so that anew optical detection of floating DNA may be carried out. However, inthis case information related to probe location and type would be lost,so different species of amplified target DNA cannot be distinguished atthis stage of the cycle. However, the signal will be diffused throughoutthe entire solution, and be less intense that the localized signal thatoccurs when the localized probes hybridize to target.

During each cycle, the heating rate is preferably at least 5-7° C./s,while the cooling rate is preferably greater than 10° C./s.

The hybridization and annealing/amplification cycles may be repeateduntil sufficient copies of the target DNA have been produced as to bedetectable. The amplification process may then be interrupted uponpositive detection of the searched target DNA, or after a thresholdnumber of cycles, if the target DNA is not detected (it is thusdetermined that the starting sample did not contain the target DNA).

Thus, real-time detection of multiple target DNA is possible, becausehybridization of the probes and primer annealing take place at separatetemperatures. In turn, real-time detection allows the operator to reducethe average duration of amplification processes, because amplificationmay be interrupted as soon the amount of target DNA becomes detectable.Moreover, use of a microarray of probes allows the operator tosimultaneously detect the presence or absence of numerous differenttarget DNA through a single fluorescent dye. In fact, different probesare usually provided at different locations in the microarray, thus thelocation at which hybridization of a probe is detected carries alsoinformation on the nature of the target DNA bound to the probe.

Finally, it is clear that numerous modifications and variations may bemade to the method and apparatus described and illustrated herein, allfalling within the scope of the invention, as defined in the attachedclaims.

1. Method for carrying out nucleic acid amplification, comprising:providing a reaction chamber, accommodating an array of nucleic acidprobes at respective locations, for hybridizing to respective targetnucleic acids; and introducing a solution into the reaction chamber,wherein the solution contains primers, capable of binding to targetnucleic acids, nucleotides, nucleic acid extending enzymes and nucleicacids; characterized by selecting a structure of the nucleic acid probesand of the primers so that a hybridization temperature (TH) of theprobes is higher than an annealing temperature (TA) of the primers andhybridization and annealing take place in respective separatetemperature ranges (R_(H), R_(A)).
 2. Method according to claim 1,comprising cyclically controlling an operative temperature (T_(O)) ofthe solution in accordance with a temperature profile.
 3. Methodaccording to claim 2, wherein the separate temperature ranges (R_(H),R_(A)) comprise a hybridization temperature range (R_(H)) and anannealing temperature range (R_(A)) and wherein cyclically controllingthe operative temperature (T_(O)) comprises setting the solution to afirst temperature (T_(H)) within the hybridization temperature range(R_(H)), in which hybridization of the probes is allowed, and settingthe solution to a second temperature (T_(A)) within the annealingtemperature range (R_(H)), in which annealing of the primers is allowed.4. Method according to claim 3, wherein the annealing temperature range(R_(A)) is lower than the hybridization temperature range (R_(H)). 5.Method according to claim 4, comprising carrying out detection ofhybridized probes between setting the solution to the first temperature(T_(H)) and setting the solution to the second temperature (T_(A)). 6.Method according to claim 5, comprising terminating amplification whenat least one of a first condition and a second condition is met, whereinthe first condition includes positive detection of hybridized probes andthe second condition includes carrying out a threshold number ofamplification cycles.
 7. Method according to claim 6, wherein cyclicallycontrolling comprises: first setting the solution to a third temperature(T_(D)), higher than the hybridization temperature range (R_(H)), todenature the nucleic acids before setting the solution to the firsttemperature (T_(H)); and second setting the solution to the thirdtemperature (T_(D)), to denature the nucleic acids between setting thesolution to the first temperature (T_(H)) and setting the solution tothe second temperature (T_(A)).
 8. Method according to claim 7,comprising setting the solution to a fourth temperature (TE),intermediate between the first temperature (TH) and the secondtemperature (TA) and such that extension of primers is allowed.
 9. Amicroreactor comprising: a reaction chamber, accommodating an array ofnucleic acid probes at respective locations, for hybridizing torespective target nucleic acids; and a solution in the reaction chamber,wherein the solution contains primers, capable of binding to targetnucleic acids, nucleotides, nucleic acid extending enzymes and nucleicacids; characterized in that the nucleic acid probes and the primers arestructured so that a hybridization temperature (T_(H)) of the probes ishigher than an annealing temperature (T_(A)) of the primers andhybridization and annealing take place in respective separatetemperature ranges (R_(H), R_(A)).
 10. (canceled)
 11. A microreactoraccording to claim 9, further comprising a temperature control modulefor cyclically controlling an operative temperature (T_(O)) of thesolution in accordance with a temperature profile.
 12. A microreactoraccording to claim 11, wherein separate temperature ranges (R_(H),R_(A)) comprise a hybridization temperature range (R_(H)) and anannealing temperature range (R_(A)) and wherein the temperature controlmodule is configured to set the solution to a first temperature (T_(H))within the hybridization temperature range (R_(H)), in whichhybridization of the probes is allowed, and to set the solution to asecond temperature (T_(A)) within the annealing temperature range(R_(H)), in which annealing of the primers is allowed.
 13. Amicroreactor according to claim 11, wherein the temperature controlmodule comprises a temperature controller, a power source and a coolingelement, both controlled by the temperature controller and operable torespectively heat and cool the microreactor in accordance with thetemperature profile.
 14. A microreactor according to claim 12, furthercomprising a reader device and wherein the microreactor is mounted on aboard to form a cartridge loadable into the reader device.
 15. Amicroreactor according to claim 14, wherein the reader device is anoptical reader.
 16. A microreactor according to claim 15, wherein thereader device includes: a light source for illuminating the microreactorwith light at an excitation wavelength, when the cartridge is loaded inthe reader device; and an image detector, configured to receivefluorescence radiation emitted the microreactor, in response to thelight at the excitation wavelength.